(a) Field of the invention
The present invention relates to the production of paclitaxel and derivatives thereof (such as related taxanes) using a plurality of different bacteria isolated from different species of Taxus, and also to a novel taxane. There are disclosed methods for the isolation of these bacteria and the screening tests that were used to provide evidence that paclitaxel and taxanes were produced by said bacteria. There are also disclosed methods for the biotransformation of pro-taxanes by said bacteria.
(b) Description of prior art
Paclitaxel, also referred to as Taxol.TM., has been first identified in 1971 by Wani and collaborators (Wani MC et al., 1971 J. Am. Chem. Soc., 93: 2325-2327) following a screening program of plant extracts of the National Cancer Institute. This complex diterpene showed cytotoxic activity against several types of tumors and is presently used in the treatment of some cancers such as ovarian and breast cancers. Clinical studies suggest that Taxol.TM. could eventually be used in the treatment of over 70% of human cancers.
Paclitaxel differs from other cytotoxic drugs by its unique mechanism of action. It interferes with cell division by manipulating the molecular regulation of the cell cycle. Paclitaxel binds to tubulin, the major structural component of microtubules that are present in all eukaryotic cells. Unlike other antimitotic agents such as vinca alkaloids and colcichine, which inhibit the polymerization of tubulin, paclitaxel promotes this assembly of tubulin and stabilizes the resulting microtubules. This event leads to the interruption of cell division, and ultimately to cell death.
The major obstacle in the use of paclitaxel as an anticancer treatment is its supply. It was originally isolated from the bark and leaves of yew trees such as Taxus brevifolia, T. baccata, T. cuspidata or, T. canadensis. The low yield of the isolation of paclitaxel (0.016 g %) and the limited availability of the trees have forced the scientific and industrial community to find alternative ways of producing paclitaxel.
The antitumor property of taxoid compounds has also lead to the generation of new anticancer drugs derived from taxanes. Taxotere.TM. (sold by Rhone-Poulenc Rorer), which is produced from 10-deacetylbaccatin III by hemisynthesis, is currently used in the treatment of ovarian and breast cancers. Furthermore, Abbott Laboratories is conducting clinical trials with a drug derived from 9-dihydro-13-acetyl baccatin III, a natural precursor specific to Taxus canadensis. The increasing demand for taxanes highlights the urgent need for renewable and economical processes that would not endanger plant species.
Presently, industrials are producing paclitaxel through hemisynthesis from baccatin III, a natural precursor of paclitaxel. However, this process still relies on a plant substance that must be extracted from yew trees. The first complete chemical synthesis of paclitaxel has been achieved in 1994 by Nicolaou et al. (1994, Nature, 367:630-634). This is a multistep process and the overall yield has made this approach non economically feasible.
Plant cell culture of Taxus species is another approach explored by many groups (Yukimune et al., 1996, Nature Biotechnology, 14:1129-1132; Srinivasan et al., 1995, Biotechnology and Bioengineering, 47:666-676). Somehow, this process is limited by the amount of paclitaxel that can be produced, the length of incubation time required to obtain significant yields, and the application of plant cell culture to the large volumes required by the industry.
In U.S. Pat. No. 5,322,779, in the names of Gary A. Strobel et al. disclosed a fungus isolated from the bark of a sample of Taxus brevifolia which is able to synthetize paclitaxel at a level of 24-50 ng/l after a period of incubation of 3 weeks. Later, Strobel et al. (1996, Microbiology, 142:435-440) reported another fungus, Pestalotiopsis microspora, isolated from the inner bark of Taxus wallachiana that can produced up to 55 .mu.g/l of paclitaxel within 5 weeks. Somehow, the long periods of incubation and the large volumes required to extract significant amounts of paclitaxel reduce the profitability of the process.
In U.S. Pat. No. 5,561,055 (issued on Oct. 1, 1996 in the names of Michel Page et al., the Applicant), there is disclosed one bacterium, which was referred to as Erwinia taxi, for the production of paclitaxel. Since then, this bacteria has been characterized as Sphingomonas taxi. This bacterium was isolated from Taxus canadensis. It would be highly desirable to be provided with other bacteria having highly diverse metabolic capacities isolated from different species of Taxus for the production of paclitaxel and related taxanes at higher yields.
It would also be highly desirable to be provided with widely different bacteria for the mass production of various different bacterial taxanes.
As mentioned in International Patent Application published under number W097/16200, biotransformation process may be used for the generation of new taxanes molecules that lead to new therapeutic drugs. It would also be highly desirable to be provided with new strains of microorganisms able to biotransform taxanes compounds for use as therapeutic agents or to be modified by hemisynthesis.
Genetic manipulations of bacteria can increase the activity or the production of certain proteins. It would also be highly desirable to be provided with mutant of our original isolates that could produce and biotransform taxanes at higher levels.